Metal ions play an important role in biological systems in regulating enzyme activity, protein structure, and cellular signaling. Metal ions can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete. A large number of natural and synthetic materials are known to selectively or non-selectively bind to or chelate metal ions. Ion chelators are commonly used in solution for in vivo control of ionic concentrations and detoxification of excess metals, and as in vitro buffers. When bound to a fluorophore, ion chelators can often be used as optical indicators of ionic transients.
The detection and quantification of calcium ion (Ca.sup.2+) levels in biological systems, in particular, has become an important area of investigation in biological and medical research. The most reliable indicators for Ca.sup.2+ ions to date utilize a tetracarboxylate chelating group based upon the structure of 1,2-bis-2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA), usually in conjunction with a covalently attached fluorophore. Upon binding Ca.sup.2+ in the BAPTA chelate of the indicator, the fluorescence properties of the attached fluorophore are generally affected in some measurable way (i.e. emission is enhanced or decreased, the wavelength of excitation or emission is altered, etc.). Once the dissociation constant for a specific indicator-Ca.sup.2+ complex is known, a measurement of the fluorescence properties of a sample containing the indicator allows a determination of in situ Ca.sup.2+ concentration (Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Set 20 (1992)).
Examples of BAPTA-based fluorescent indicators known in the art include quin-2 (Tsien, BIOCHEMISTRY 19, 2396 (1980)); fura-2, and indo-1 (U.S. Pat. No. 4,603,209 to Tsien et al., 1986); fluo-3 and rhod-2 (U.S. Pat. No. 5,049,673 to Tsien et al. 1991, incorporated by reference); and FURA-RED.TM. (Molecular Probes, Inc., Eugene, Oreg., trademark for 1- 6-amino-2-(5-oxo-2-thioxo-4-thiazolidinylidene)methyl-5-benzofuranyloxy !-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid and the tetraacetoxymethyl ester thereof, U.S. Pat. No. 4,849,362 to DeMarinis, et al. 1989). Additional BAPTA-based fluorescent indicators for Ca.sup.2+ have been described by Tsien (Intracellular Measurements of Ion Activities ANN. REV. BIOPHYS. BIOENG., 12, 91 (1983)), and Smith et al., (J. CHEM. SOC. PERKIN TRANS. 2, 1195 (1993)) however these indicators all have deficiencies in fluorescent response or other properties, thereby limiting their utility.
The indicators fura-2, indo-1 and quin-2 share a common drawback in that the excitation bands for these indicators occur at short wavelengths (340-360 nm). Radiation at these high energy wavelengths has been shown to damage cellular structures, as well as interfere with detection of the resulting fluorescence due to the natural fluorescence many biological materials exhibit at these wavelengths.
The related indicators fluo-3 and rhod-2, containing a fluorescein and rhodamine fluorophore, respectively, were designed to overcome this limitation. These indicators may be used at much longer wavelengths, eliminating the difficulties created by UV irradiation. In addition, these indicators exhibit enhanced fluorescence upon Ca.sup.2+ binding, simplifying the analysis of Ca.sup.2+ concentration. The ability to use longer wavelength light both for excitation and emission means that conventional optics and filters can be used in Ca.sup.2+ determination (in conjunction with flow cytometry or fluorescence microscopy, for example) rather than the specialized and expensive quartz optics required for UV analysis.
It is frequently desirable to have a selection of indicators having a wide range of spectral responses so as to make simultaneous or sequential determination of several analytes, or to be able to demonstrate labeling of spatially resolved sites in biological cells. A novel series of BAPTA-type indicators have been prepared with reactive sites that allow the attachment of a wide range of fluorophores as well as polymolecular assemblies or lipophilic moieties (Copending application REACTIVE DERIVATIVES OF BAPTA USED TO MAKE ION-SELECTIVE CHELATORS, Ser. No. 07/843,360, now U.S. Pat. No. 5,453,517, (filed Feb. 25, 1992) to Kuhn et al., incorporated by reference). By attachment to organelle-targeting peptides, polymeric conjugates of Ca.sup.2+ indicators have been targeted to cellular organelles, including the nucleus (Copending application BIFUNCTIONAL CHELATING POLYSACCHARIDES, Ser. No. 08/082,269 (filed Jun. 23, 1993) to Kuhn et al., incorporated by reference).
While previous Ca.sup.2+ indicators, such as those described above, have generally been designed to maximize metal ion affinity, under some circumstances a high binding affinity is undesirable. An ion indicator will have its maximum sensitivity in the range of its dissociation constant for the ion. When the concentration of the ion is more than one log unit above the dissociation constant for the metal, the indicator becomes saturated and unresponsive to further increases in the ion's concentration. For example, a commonly used fluorescent Ca.sup.2+ indicator, fura-2, with a Ca.sup.2+ dissociation constant of 224 nM, cannot be used to measure Ca.sup.2+ levels about 1 .mu.M. In situations where high physiological concentrations of Ca.sup.2+ are present (as in gap junctions or in some extracellular spaces), indicators with a lower affinity are desirable to ensure a measurable response.
A currently available Ca.sup.2+ indicator that possesses a lower binding affinity, CALCIUM GREEN.TM.-5N (Molecular Probes, Inc., Eugene, Oreg.) suffers from the drawback that Ca.sup.2+ binding to the indicator results in an increase in emission intensity only. The indicator displays no wavelength shift in either the excitation or emission spectrum upon binding, which makes the concentration of Ca.sup.2+ difficult to measure using conventional ratiometric techniques (infra).
The compounds of the present invention, which are based on the benzazolylcoumarin fluorophore, exhibit absorbance and emission that is typically at wavelengths between those of the UV-excited indicators and indicators such as fluo-3, rhod-2, CALCIUM GREEN.TM. and FURA-RED.TM.. However the compounds of the present invention retain the desirable property of a shift in excitation bands upon binding to metal ions, which allows Ca.sup.2+ detection or quantification by ratiometric analysis of the excitation spectra of the indicator.
The compounds of the present invention have significant utility as a means of detecting and quantifying metal cation levels in living cells, biological fluids or aqueous solutions. The long wavelength excitation and emission bands of the compounds of the present invention enable their use with a variety of optical devices and require no specialized (quartz) optics, such as are required by indicators which are excited or emit at shorter wavelengths. In addition, the generally weaker affinity of the compounds of the present invention allow the quantification of metal ion levels that are too high for measurement by previously available metal ion indicators that are excited at similar or shorter wavelengths, which simply saturate at higher concentrations. These indicators are suitable for use in fluorescence microscopy, flow cytometry, fluoroscopy, or any other application that currently utilize fluorescent metal ion indicators.